CN110431153B

CN110431153B - PCSK-9 antibody pharmaceutical composition and application thereof

Info

Publication number: CN110431153B
Application number: CN201880019136.3A
Authority: CN
Inventors: 田晨敏; 李皓; 刘洵
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd; Suzhou Suncadia Biopharmaceuticals Co Ltd
Current assignee: Guangdong Jiangsu Hengrui Medicine Co ltd; Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd; Suzhou Suncadia Biopharmaceuticals Co Ltd
Priority date: 2017-06-30
Filing date: 2018-06-29
Publication date: 2023-09-19
Anticipated expiration: 2038-06-29
Also published as: CN110431153A; CA3067953A1; EP3647325A4; RU2020102952A3; US20200223941A1; EP3647325A1; TWI720325B; WO2019001560A1; RU2020102952A; AU2018291702A1; JP2020532491A; TW201904609A

Abstract

The application provides a pharmaceutical composition comprising a PCSK9 antibody or antigen-binding fragment thereof in a histidine buffer. In addition, the pharmaceutical composition may contain sugar and a nonionic surfactant.

Description

PCSK-9 antibody pharmaceutical composition and application thereof

The present application claims priority from chinese patent application CN201710519829.6, whose filing date is 2017, 6, 30. The present application incorporates the entirety of the above-mentioned chinese patent application.

Technical Field

The application belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition containing a PCSK-9 antibody and an antigen binding fragment thereof, and application of the pharmaceutical composition as a medicament.

Background

Hypercholesterolemia is a disorder of lipid metabolism characterized mainly by elevated serum cholesterol levels, which is mainly manifested by elevated serum cholesterol levels, leading to aggregation of cholesterol in blood vessels, forming atherosclerosis. A large number of clinical and experimental research results prove that the lipid metabolism abnormality and the occurrence and development of coronary heart disease have close relation. Therefore, lowering the concentration of cholesterol in the blood is currently a major means of treating and preventing atherosclerosis.

Along with the rapid improvement of the national living standard of China, dyslipidemia also becomes a main factor for endangering urban and rural residents of China. According to 2012 statistics, about 40% of deaths in our country die from cardiovascular disease. At present, the prevalence rate of the dyslipidemia of adults in China is 18.6%, and the estimated number of people suffering from the national dyslipidemia is 1.6 hundred million. The occurrence rate of different types of dyslipidemia is respectively as follows: hypercholesterolemia 2.9%, hypertriglyceridemia 11.9%, low high density lipoproteinemia 7.4%; an additional 3.9% of human blood cholesterol had marginally raised. The medical science of "chronic disease control Chinese expert consensus" reached by the Committee of the department of health of disease control expert, the department of health, the disease control expert, mentions that there are 3300 ten thousand hypercholesterolemia patients in China, but the incidence of dyslipidemia in China is far worse than the above data from the local area.

Currently, clinically lipid-regulating drugs mainly comprise statins. Liptol (Liptr) is the most widely used cholesterol-lowering drug worldwide, and is the most popular drug in medical history, and by blocking the enzyme action of liver to produce cholesterol, the production of cholesterol is reduced, so that the liver can take more cholesterol from blood, and the concentration of cholesterol in blood is reduced. However, there are disadvantages to the use of lipstatin, in that firstly, from the data, lipstatin can reduce 30% -40% of low density lipoproteins, but still many patients still cannot reach an effective blood lipid reduction (low density lipoproteins concentration <50 mg/dL), and secondly, the response rate of the patients to the lipstatin is also humanoid. For these reasons, a more effective hypolipidemic agent is needed for patients.

Familial hypercholesterolemia (Familial hypercholesterolemia, FM) is an autosomal monogenic dominant inherited disease characterized clinically by significant elevation of total blood cholesterol and low density lipoprotein cholesterol (low density lipoprotein-cholesterol, LDL-c), blepharofloxama, corneal arcus, and early-onset cardiovascular disease. The mutation of Low Density Lipoprotein Receptor (LDLR) gene causes its deficiency or deficiency, and LDL-c is not smoothly transported to liver for clearance, so that the level of LDL-c in blood is increased. It has been clarified that 3 genes are involved in the occurrence of FM, which are the LDLR gene, the apolipoprotein B100 gene and the PCSK9 (proprotein convertase subtilisin/kexin type 9) gene, respectively.

Proprotein convertase subtilisin 9 (PCSK 9) is a proprotein convertase belonging to the proteinase K subfamily of the secreted subtilase family. The encoded protein is synthesized as a soluble zymogen and undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. Experimental results show that PCSK9 promotes the degradation of LDL receptors to increase LDL cholesterol levels in plasma, while LDL receptors mediate the process of LDL endocytosis in the liver, the latter being the primary pathway for the clearance of LDL from the circulatory system. It was found that 12.5% of patients with hypercholesterolemia (ADH) were detected with PCSK9 gene mutation. PCSK9 is diverse in mutant forms and can be classified into two categories based on the different effects of mutation on PCSK9 regulation of LDL-C levels: a loss-of-function type and a gain-of-function type. Wherein the loss-of-function mutation is associated with low blood cholesterol levels, has the effect of preventing coronary atherosclerotic heart disease from occurring, and has a higher mutation rate of PCSK9 with low cholesterol than other ethnicities in African population. PCSK9 function-acquired mutants increase plasma cholesterol levels by increasing PCSK9 function, decreasing LDLR expression, which can lead to severe hypercholesterolemia and early coronary atherosclerotic heart disease, the PCSK9 function-acquired mutations currently found include: D374Y, S127R, F216L, N157K, R306S, etc. Wherein, compared with the wild type PCSK9, the LDLR on the cell surface of the D374Y mutant is reduced by 36 percent, and the S127R mutation is correspondingly reduced by 10 percent.

However, antibody drugs have a large molecular weight and a complex structure, and are easily degraded, polymerized, or subjected to undesired chemical modification, and thus are unstable. In order to make antibodies suitable for administration and maintain stability during storage and subsequent use, the study of stable formulations of antibody drugs is particularly important.

Although, there are several companies currently developing pharmaceutical formulations comprising PCSK-9 antibodies, such as: CN103717237A, CN104364266a, etc. There is still a need for new PCSK-9 antibodies to develop pharmaceutical (formulation) compositions comprising PCSK-9 that are more suitable for administration.

Disclosure of Invention

The present invention provides a pharmaceutical composition comprising a PCSK-9 antibody or antigen-binding fragment thereof, and a buffer, preferably a histidine or succinate or phosphate or citrate buffer, more preferably a histidine buffer, most preferably a histidine-hydrochloride buffer.

In alternative embodiments, the pharmaceutical composition wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of about 1mg/ml to 150mg/ml, preferably about 30mg/ml to 100mg/ml, most preferably 50-60mg/ml, more preferably 50mg/ml. Non-limiting examples include 45mg/ml, 46mg/ml, 47mg/ml, 48mg/ml, 49mg/ml, 50mg/ml, 51mg/ml, 52mg/ml, 53mg/ml, 54mg/ml, 55mg/ml, 56mg/ml, 57mg/ml, 58mg/ml, 59mg/ml, 60mg/ml.

In alternative embodiments, the concentration of buffer is about 5mM to 50mM, preferably about 5mM to 30mM, non-limiting examples include 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, more preferably 10mM to 15mM, and most preferably 10mM.

In alternative embodiments, the pH of the buffer in the pharmaceutical composition is about 5.5 to 6.5, preferably about 6.0 to 6.5, non-limiting examples include about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5.

Further, in an alternative embodiment, the pharmaceutical composition further comprises a sugar. The "sugar" of the present invention comprises a conventional composition (CH ₂ O) _n And derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. Can be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannose, stachyose, maltose, lactulose, maltose, sorbitol, maltitol, lactitol, isomalt, etc. The preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose.

In alternative embodiments, the concentration of sugar in the pharmaceutical composition is from about 10mg/ml to about 75mg/ml, preferably from 20mg/ml to about 60mg/ml, more preferably from 20mg/ml to about 40mg/ml, most preferably 25mg/ml, non-limiting examples include 20mg/ml, 21mg/ml, 22mg/ml, 23mg/ml, 24mg/ml, 25mg/ml, 26mg/ml, 27mg/ml, 28mg/ml, 29mg/ml, 30mg/ml, 31mg/ml, 32mg/ml, 33mg/ml, 34mg/ml, 35mg/ml, 36mg/ml, 37mg/ml, 38mg/ml, 39mg/ml, 40mg/ml.

In an alternative embodiment, the pharmaceutical composition further comprises a surfactant. May be selected from polysorbate 20, polysorbate 80, polyhydroxyen, triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl-, myristyl-, linoleyl-, stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, myristyl-, cetyl-betaine, lauramidopropyl-, cocamidopropyl-, oleamidopropyl-, myristamidopropyl-, palmamidopropyl-, isostearamidopropyl-betaine, myristamidopropyl-, palmamidopropyl-, isostearamidopropyl-dimethylamine, sodium methyl cocoa acyl, sodium methyl oleyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, and the like. The preferred surfactant is polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

In alternative embodiments, the concentration of surfactant in the pharmaceutical composition is from about 0.05mg/ml to 1.0mg/ml, preferably from 0.1mg/ml to 0.4mg/ml, non-limiting examples include 0.1mg/ml, 0.15mg/ml, 0.2mg/ml, 0.25mg/ml, 0.3mg/ml, 0.35mg/ml, 0.4mg/ml, further preferably from 0.1mg/ml to 0.3mg/ml, more preferably from 0.1mg/ml to 0.2mg/ml, and most preferably 0.2mg/ml.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 1-150mg/ml of a PCSK-9 antibody or antigen-binding fragment thereof,

(b) 5-30mM histidine buffer, pH about 5.5-6.5, more preferably about 6.0-6.5

(c) Sucrose 10-75mg/ml, and

(d) 0.05-0.6mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 50mg/ml PCSK-9 antibody or antigen-binding fragment thereof;

(b) 5-20mM histidine buffer;

(c) 25mg/ml sucrose; and

(d) 0.1-0.3mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 1-150mg/ml PCSK-9 antibody or antigen-binding fragment thereof, and

(b) 5-30mM histidine buffer;

and the pharmaceutical composition has a pH of about 6.0 to about 6.5.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 1-150mg/ml PCSK-9 antibody or antigen-binding fragment thereof;

(b) 5-30mM histidine buffer;

(c) Sucrose 10-75 mg/ml; and

(d) 0.05-0.6mg/ml polysorbate 80 and the pharmaceutical composition has a pH of about 6.0-6.5. The histidine buffer is preferably histidine-hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 50mg/ml PCSK-9 antibody or antigen-binding fragment thereof;

(b) 5-20mM histidine buffer;

(c) 25mg/ml sucrose; and

(d) 0.1-0.3mg/ml polysorbate 80 and the pharmaceutical composition has a pH of about 6.0-6.5. The histidine buffer is preferably histidine-hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 50mg/ml PCSK-9 antibody or antigen-binding fragment thereof;

(b) 10mM histidine buffer;

(c) 25mg/ml sucrose; and

(d) 0.2mg/ml polysorbate 80 and the pH of the pharmaceutical composition was 6.3±0.1. The histidine buffer is preferably histidine-hydrochloride buffer.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 150mg/ml PCSK-9 antibody or antigen-binding fragment thereof; (b) 30mM histidine-hydrochloride buffer; (c) 75mg/ml sucrose; and (d) 0.6mg/ml polysorbate 80; the final pH of the pharmaceutical composition was 6.3.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 50mg/ml PCSK-9 antibody or antigen-binding fragment thereof; (b) 10mM histidine-hydrochloride buffer, pH 6.0; (c) 25mg/ml sucrose; and (d) 0.2mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 150mg/ml PCSK-9 antibody or antigen-binding fragment thereof; (b) 20mM histidine-hydrochloride buffer, pH 6.5; and (c) 70mg/ml sucrose.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 150mg/ml PCSK-9 antibody or antigen-binding fragment thereof; (b) 20mM histidine-hydrochloride buffer, pH 6.5; and (c) 70mg/ml of alpha, alpha-trehalose dihydrate.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 50mg/ml PCSK-9 antibody or antigen-binding fragment thereof; (b) 20mM histidine-hydrochloride buffer, pH 6.0; (c) 25mg/ml sucrose; and (d) 0.2mg/ml polysorbate 80.

In an alternative embodiment, the pharmaceutical composition comprises:

(a) 50mg/ml PCSK-9 antibody or antigen-binding fragment thereof; (b) 20mM histidine-hydrochloride buffer, pH 6.5; (c) 25mg/ml sucrose; and (d) 0.2mg/ml polysorbate 80.

In an alternative embodiment, the PCSK-9 antibody or antigen-binding fragment in the pharmaceutical composition has the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and

LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17, respectively.

In alternative embodiments, the antibody or antigen binding fragment in the pharmaceutical composition may be selected from murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

In alternative embodiments, the light chain of the PCSK-9 antibody has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the antibody light chain amino acid sequence of h001-4-YTE in a pharmaceutical composition, the heavy chain amino acid sequence of the PCSK-9 antibody has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the antibody heavy chain of h001-4-YTE, the h001-4-YTE antibody light chain sequence being as set forth in SEQ ID NO:30, the heavy chain sequence of the h001-4-YTE antibody is shown as SEQ ID NO: shown at 32.

The present invention also provides a method for preparing the aforementioned pharmaceutical composition, comprising: a step of replacing the stock solution of the PCSK-9 antibody or an antigen-binding fragment thereof with a buffer, preferably a histidine buffer, more preferably a histidine-hydrochloride buffer, at a concentration of preferably about 5mM to 30mM, non-limiting examples of which include 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, more preferably 10mM to 15mM; the buffer pH is about 6.0 to 6.5, non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.

Stock solution further, the method of preparing the aforementioned pharmaceutical composition, further comprising the step of adding sucrose and polysorbate 80 to the solution obtained in the displacement step, and sizing the solution with a buffer, wherein the buffer concentration is preferably about 10mM to 20mM, non-limiting examples include 10mM, 12mM, 14mM, 16mM, 18mM, 20mM; the buffer pH is about 6.0 to 6.5, non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.

The invention also provides a method for preparing a freeze-dried preparation containing the PCSK-9 antibody, which comprises the step of freeze-drying the pharmaceutical composition.

In an alternative embodiment, the freeze-drying in the method of preparing a lyophilized formulation comprising a PCSK-9 antibody comprises the steps of pre-freezing, primary drying and secondary drying in that order. The pre-freezing is from 5 ℃ to-40 ℃ to-50 ℃, and is optimally-45 ℃, and the vacuum degree is not required. The primary drying temperature is between-14 ℃ and 0 ℃, and is optimally between-5 ℃; the vacuum degree is 0.1mBar-0.5mBar, and the optimal vacuum degree is 0.3mBar. The secondary drying temperature is 20-30 ℃, optimally 25 ℃, and the vacuum degree is reduced from 0.1mBar to 0.5mBar, optimally 0.3mBar, to 0.005mBar to 0.02mBar, optimally 0.01mBar.

The invention also provides a freeze-dried preparation containing the PCSK-9 antibody, which is prepared by the method for preparing the freeze-dried preparation containing the PCSK-9 antibody.

In some embodiments, the lyophilized formulation is stable at 2-8 ℃ for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

The present invention also provides a method of preparing a reconstituted solution of a lyophilized formulation comprising a PCSK-9 antibody, comprising the step of reconstituting the lyophilized formulation as described above, with a solution selected from, but not limited to, water for injection, physiological saline or dextrose solution.

The invention also provides a re-dissolving solution of the freeze-dried preparation containing the PCSK-9 antibody, which is prepared by the method for preparing the re-dissolving solution of the freeze-dried preparation containing the PCSK-9 antibody.

In an alternative embodiment, the PCSK-9 antibody-containing reconstituted solution of the invention contains 2-5 times, preferably 3 times the concentration of the components of the pre-lyophilized pharmaceutical composition.

In alternative embodiments, the reconstituted solution containing the PCSK-9 antibody, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of about 120mg/ml to 200mg/ml, most preferably 150mg/ml.

In an alternative embodiment, the reconstituted solution containing PCSK-9 antibody, wherein the pharmaceutical composition has a pH of about 6.0 to about 6.5, preferably about 6.4. The pH of the reconstituted solution is related to the pH of the buffer of the stock solution used for preparation, and when the pH of the stock solution is 6.0, the pH of the final reconstituted solution is 6.3+/-1.

In an alternative embodiment, the reconstitution solution containing the PCSK-9 antibody, wherein the buffer concentration is about 15mM to 45mM, preferably 30mM.

In an alternative embodiment, the PCSK-9 antibody-containing reconstituted solution further comprises a disaccharide, preferably selected from trehalose or sucrose, most preferably sucrose.

In an alternative embodiment, the reconstituted solution containing PCSK-9 antibody, wherein the sugar concentration is about 55mg/ml to 95mg/ml, preferably 75mg/ml.

In an alternative embodiment, the PCSK-9 antibody-containing reconstituted solution further comprises a surfactant, preferably polysorbate, more preferably polysorbate 80.

In an alternative embodiment, the reconstituted solution containing PCSK-9 antibody, wherein the concentration of surfactant is from about 0.4mg/ml to 0.8mg/ml, preferably 0.6mg/ml.

The invention further provides an article of manufacture or kit comprising a container containing any of the stable pharmaceutical compositions described herein. In some embodiments, the vial is a neutral borosilicate glass tube injection vial.

The invention also provides the use of a pharmaceutical composition or lyophilized formulation or a reconstituted solution of a lyophilized formulation as described above for the manufacture of a medicament for the treatment of a PCSK-9 related disease or disorder, preferably a cholesterol related disease, more preferably hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, alzheimer's disease and dyslipidemia in general, most preferably hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.

The present invention also provides a method of the former type for the treatment and prophylaxis of PCSK-9-related diseases or conditions comprising administering to a patient in need thereof a therapeutically effective amount of the aforementioned pharmaceutical composition or lyophilized formulation or reconstituted solution of lyophilized formulation, wherein the disease is preferably a cholesterol-related disease, more preferably hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, alzheimer's disease and dyslipidemia in general, most preferably hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease.

The present invention also provides an article of manufacture comprising a container containing a pharmaceutical composition or lyophilized formulation or reconstituted solution of a lyophilized formulation as described above.

It is to be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention are further described by the following detailed description.

Detailed Description

1. Terminology

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

"buffer" refers to a buffer that is resistant to pH changes by the action of its acid-base conjugated components. Examples of buffers to control the pH in the appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine and other organic acid buffers.

A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and like buffers, preferably histidine-hydrochloride buffers. The histidine-hydrochloride buffer solution is prepared from histidine and hydrochloric acid or histidine and histidine hydrochloride.

A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

A "succinate buffer" is a buffer that includes succinate ions. Examples of succinate buffers include sodium succinate, potassium succinate, calcium succinate and the like. The preferred succinate buffer is sodium succinate-succinate.

A "phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like. The preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

An "acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate.

"pharmaceutical composition" means a mixture comprising one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredients of the antibody, promote the administration to organisms, and facilitate the absorption of the active ingredients so as to exert biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not intended to be mutually exclusive.

By "lyophilized formulation" is meant a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a liquid or solution formulation.

The freeze-drying of the invention comprises pre-freezing, primary drying and secondary drying. Prefreezing is the purpose of freezing a product to obtain a crystalline solid. The pre-freezing temperature and the pre-freezing speed are two important technological parameters, and the pre-freezing temperature is set to be-45 ℃ and the pre-freezing speed is set to be 1 ℃/min. Primary drying, also known as primary drying, is the primary stage of sample freeze drying. The purpose is to remove ice in the product while maintaining the shape of the product and minimizing damage to the product. If the temperature and vacuum of the primary drying are selected improperly, this can result in collapse of the article. Higher temperatures and vacuum levels increase lyophilization efficiency, but at the same time increase the risk of product collapse. The temperature of the primary drying according to the invention may be conventional in the art, for example-27 ℃ to 0 ℃, preferably-14 ℃ to-5 ℃. Secondary drying, also known as resolution drying, is the main step of removing the bound water from the product by pulling a final vacuum (0.01 mbar) and elevated temperature (20-40 ℃). Since most biologicals are relatively sensitive to temperature, the secondary drying temperature is chosen at the low point of the temperature range, i.e. 25 ℃. The time of lyophilization is related to the freezer, the dose of lyophilized formulation, and the container of lyophilized formulation. Such time adjustments are well known to those skilled in the art.

The pharmaceutical composition provided by the invention can achieve a stable effect: a pharmaceutical composition wherein the antibody substantially retains its physical and/or chemical stability and/or biological activity after storage, preferably the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are a number of analytical techniques for measuring protein stability that measure stability after storage at a selected temperature for a selected period of time.

A stable pharmaceutical antibody formulation is one in which no significant change is observed in the following cases: the storage is at refrigeration temperature (2-8 ℃) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, stable liquid formulations include those that: which exhibits desired characteristics after storage at temperatures including 25 ℃ and 40 ℃ for periods including 1 month, 3 months, 6 months. Typical acceptable criteria for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of the antibody monomers degrade as measured by SEC-HPLC. The pharmaceutical antibody formulation was colorless, or clear to slightly milky, by visual analysis. The concentration, pH and osmolality of the formulation have a variation of no more than + -10%. Typically no more than about 10%, preferably no more than about 5% truncation is observed. Usually no more than about 10%, preferably no more than about 5% of aggregates are formed.

An antibody "retains its physical stability" in a pharmaceutical formulation if it does not exhibit a significant increase in aggregation, precipitation and/or denaturation after visual inspection of color and/or clarity, or as measured by UV light scattering, size Exclusion Chromatography (SEC) and Dynamic Light Scattering (DLS). The change in protein conformation can be assessed by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).

An antibody "retains its chemical stability" in a pharmaceutical formulation if it does not exhibit a significant chemical change. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often alter the chemical structure of proteins include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide spectroscopy in combination with mass spectrometry or MALDI/TOF/MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.), and isomerization (assessed by measuring isoaspartic acid content, peptide spectroscopy, etc.).

An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of biological activities exhibited when the pharmaceutical formulation is prepared. The biological activity of an antibody may be determined, for example, by an antigen binding assay.

The three-letter and one-letter codes for amino acids used in the present invention are as described in J.biol. Chem,243, p3558 (1968).

The antibody disclosed by the invention refers to an immunoglobulin, and is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through inter-chain disulfide bonds.

In the present invention, the antibody light chain of the present invention may further comprise a light chain constant region comprising a kappa, lambda chain of human or murine origin or variants thereof.

In the present invention, the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, igG2, igG3, igG4 or variants thereof of human or murine origin.

The sequences of the heavy and light chains of antibodies, near the N-terminus, vary widely, being the variable region (Fv region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region. The variable region includes 3 hypervariable regions (HVRs) and 4 Framework Regions (FR) that are relatively conserved in sequence. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in the order from amino-to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in accordance with the known Kabat numbering convention (LCDR 1-3, HCDE2-3) or with the numbering convention of Kabat and chothia (HCDR 1).

Antibodies of the invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

The term "murine antibody" is herein a monoclonal antibody to human PCSK-9 prepared according to the knowledge and skill in the art. The preparation is performed by injecting a test subject with PCSK-9 antigen, and then isolating hybridomas expressing antibodies having the desired sequence or functional properties.

The term "chimeric antibody (chimeric antibody)" refers to an antibody in which a variable region of a murine antibody is fused to a constant region of a human antibody, and which can reduce an immune response induced by the murine antibody. The chimeric antibody is established by firstly establishing a hybridoma secreting the mouse-derived specific monoclonal antibody, cloning a variable region gene from a mouse hybridoma cell, cloning a constant region gene of a human antibody according to requirements, connecting the mouse variable region gene and the human constant region gene into a chimeric gene, inserting the chimeric gene into a human vector, and finally expressing chimeric antibody molecules in a eukaryotic industrial system or a prokaryotic industrial system. In a preferred embodiment of the invention, the antibody light chain of the PCSK-9 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof. The antibody heavy chain of the PCSK-9 chimeric antibody further comprises a heavy chain constant region of human IgG1, igG2, igG3, igG4, or variants thereof. The constant region of a human antibody may be selected from the heavy chain constant region of human IgG1, igG2, igG3 or IgG4 or variants thereof, preferably comprising the heavy chain constant region of human IgG2 or IgG4, or IgG4 which is not toxic by ADCC (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation.

The term "humanized antibody (humanized antibody)", also known as CDR-grafted antibody (CDR-grafted antibody), refers to an antibody produced by grafting the CDR sequences of a mouse into the framework of the variable region of a human antibody, i.e., into the framework sequences of a different type of human germline antibody. The strong antibody variable antibody response induced by chimeric antibodies due to the large number of mouse protein components can be overcome. Such framework sequences may be obtained from public DNA databases including germline antibody gene sequences or published references. Germline DNA sequences for human heavy and light chain variable region genes can be found, for example, in the "VBase" human germline sequence database (available in Internet www.mrccpe.com.ac.uk/VBase) and in Kabat, E.A. et al, 1991, sequences of Proteins of Immunological Interest, 5 th edition. To avoid a decrease in immunogenicity while at the same time causing a decrease in activity, the human antibody variable region framework sequences may be subjected to minimal reverse or back-mutations to maintain activity. Humanized antibodies of the invention also include humanized antibodies that are further affinity matured for the CDRs by phage display.

A "PCSK-9 antibody" is an antibody that specifically binds to PCSK-9, including but not limited to the h001 series of PCSK-9 humanized antibodies in PCT/CN 2016/111053. Wherein, the h001 series PCSK-9 humanized antibody is obtained by screening mice immunized by human PCSK-9 and humanized transformation.

The term "antigen-binding fragment" as used herein refers to Fab fragments, fab 'fragments, F (ab') 2 fragments, and ScFv fragments that bind to human PCSK-9, as well as other fragments that bind to human PCSK-9 using the VH and VL regions of antibodies that bind to human PCSK-9; comprising one or more CDR regions selected from SEQ ID NO. 1 to SEQ ID NO. 2 of the antibody of the present invention. Fv fragments contain the antibody heavy and light chain variable regions, but no constant regions, and have the smallest antibody fragment with the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The two antibody variable regions may also be joined by different linkers into one polypeptide chain, known as a single chain antibody (single chain antibody) or single chain Fv (sFv). The term "binds to PCSK-9" in the present invention means capable of interacting with human PCSK-9. The term "antigen binding site" according to the invention refers to a three-dimensional spatial site on an antigen that is recognized by the present PCSK-9 antibody or antigen-binding fragment, either continuously or discontinuously.

Methods for producing and purifying antibodies and antigen binding fragments are well known in the art, such as the guidelines for antibody experimentation in Cold spring harbor, chapters 5-8 and 15. The antibodies or antigen binding fragments of the invention are engineered to incorporate one or more human FR regions into the non-human CDR regions. Human FR germline sequences can be obtained from the website http:// IMGT. Cines. FR of ImMunoGeneTics (IMGT), or from the journal of immunoglobulins, 2001ISBN012441351 by aligning IMGT human antibody variable region germline gene databases with MOE software.

The engineered antibodies or antigen binding fragments of the invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into GS expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminal site of the Fc region. Stable clones were obtained by expression of antibodies that specifically bind to human PCSK-9. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified using conventional techniques. For example, purification is performed using an A or G Sepharose FF column containing conditioned buffer. Non-specifically bound components are washed away. The bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product is either immediately frozen, e.g., -70 ℃, or lyophilized.

"conservative modifications" or "conservative substitutions or substitutions" refer to amino acids in other amino acid substituted proteins that have similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, and rigidity, etc.) such that changes can be made frequently without altering the biological activity of the protein. Those skilled in The art know that in general, single amino acid substitutions in The non-essential region of a polypeptide do not substantially alter biological activity (see, e.g., watson et al (1987) Molecular Biology of The Gene, the Benjamin/Cummings pub. Co., page 224, (4 th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.

Amino acid sequence "identity" refers to sequence similarity between two proteins or polypeptides. When a position in both comparison sequences is occupied by the same amino acid residue, e.g., if a position in both polypeptides is occupied by the same amino acid residue, then the molecules are identical at that position. Examples of algorithms suitable for determining the percent sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms, which are described in Altschul et al (1990) J.mol.biol.215:403-410 and Altschul et al (1977) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analysis is publicly available at the national center for Biotechnology information (http:// www.ncbi.nlm.nih.gov /).

"administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refers to the contact of an exogenous drug, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "administration" and "treatment" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contacting a reagent with the cell, and contacting the reagent with a fluid, wherein the fluid is in contact with the cell. "administration" and "treatment" also mean in vitro and ex vivo treatment of, for example, a cell by an agent, diagnosis, binding composition, or by another cell. "treatment" when applied to a human, veterinary or research subject refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

"treatment" means administration of an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the invention, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the patient or population being treated to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent (also referred to as a "therapeutically effective amount") effective to alleviate any particular disease symptom can vary depending on a variety of factors, such as the disease state, age, and weight of the patient, and the ability of the drug to produce a desired therapeutic effect in the patient. Whether a disease symptom has been reduced can be assessed by any clinical test method that a physician or other healthcare professional typically uses to assess the severity or progression of the symptom. Although embodiments of the invention (e.g., therapeutic methods or articles of manufacture) may be ineffective in alleviating each target disease symptom, it should be determined according to any statistical test method known in the art, such as Student t test, chi-square test, U test according to Mann and Whitney, kruskal-Wallis test (H test), jonckheere-Terpstra test, and Wilcoxon test, that the target disease symptom should be alleviated in a statistically significant number of patients.

An "effective amount" comprises an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount is also meant to be an amount sufficient to permit or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: for example, the condition to be treated, the general health of the patient, the route and dosage of administration, and the severity of the side effects. An effective amount may be the maximum dose or regimen that avoids significant side effects or toxic effects.

"Tm value" refers to the temperature at which a protein is heat denatured, i.e., the temperature at which half of the protein is unfolded, at which time the spatial structure of the protein is destroyed, so that the higher the Tm value, the higher the thermal stability of the protein.

"displacement" refers to the displacement of a solvent system that solubilizes an antibody protein, e.g., a physically manipulated high salt or hypertonic solvent system containing an antibody protein using a buffer system of a stable formulation, such that the antibody protein is present in the stable formulation. The physical means of operation include, but are not limited to, ultrafiltration, dialysis or reconstitution after centrifugation.

2. Examples and test examples

The invention is further illustrated by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the invention.

The experimental method without specific conditions noted in the embodiment of the invention is generally according to conventional conditions; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Examples

Preparation of PCSK-9 antigen and antibody

Example 1 preparation of PCSK9 antigen and protein for detection

Protein design and expression

The amino acid sequences of antigens and proteins for detection related to the invention are designed by taking UniProt Proprotein convertase subtilisin/kexin type 9 (human PCSK9, uniprot number: Q8MBP 7) as a template of the PCSK9, different tags such as his tags or peptide fragments for promoting immunity such as PADRE peptides are optionally fused on the basis of the PCSK9 protein, and the fusion fragments are cloned on a pTT5 vector (Biovector, cat#: 102762) or a pTargeT vector (promega, A1410) respectively, transiently expressed in 293 cells or stably expressed in CHO-S, and purified to obtain the antigen and the protein for detection coded by the invention.

His-tagged PCSK9: PCSK9-His6 for use in immunogenically immunized mice or detection reagents

Annotation: the cross-hatched portion is the signal peptide and the italic portion is His6-tag (6 histidine tag).

PCSK9 with PADRE peptide and His tag: PCSK9-PADRE-His6 is used as an immunogen, and the PADRE peptide can promote immunity;

/>

Annotation: the horizontal line is signal peptide, the double-line is linker, the dot-dash line is PADRE peptide, and the italic is His6-tag.

PCSK9 and his tag fusion proteins with TEV cleavage site: PCSK9-TEV-His6, N-PCSK9 (N-terminal pCSK9 domain) can be obtained by TEV cleavage as an immunogen;

annotation: the cross line is signal peptide, the double-line is TEV enzyme cutting site, and the italic is His6-tag.

PCSK9-D374Y mutein, his-tagged: PCSK9-D374Y-His6 as a detection reagent;

/>

annotation: the cross line is marked with signal peptide, and the italic part is His6-tag.

PCSK9: PCSK9 protein with inserted biotin-receiving peptide BP15 and his tag: the PCSK9-BP15-His6 is used as a detection reagent, and the BP15 peptide position can be subjected to biotin labeling in the expression process, so that in-vitro biotin labeling and possible conformational changes are avoided;

annotation: the cross-hatched portion is the signal peptide, the double-hatched portion is the biotin-receiving peptide, and the italic portion is His6-tag.

PCSK9-Y: PCSK 9D 374Y mutant protein with inserted biotin-receiving peptide and his tag: PCSK9-D374Y-BP15-His6, protein detection;

Flag-and His-tagged pCSK9 receptor protein LDLR ectodomain fragment: LDLR-ECD-Flag-His6, detection reagent

Annotation: the horizontal line is marked with signal peptide, the double-marked line is marked with Flag label, and the italic part is His6-tag;

LDLR-Fc: shortened forms of LDLR ectodomain fragments with hIgG1-Fc fusion proteins (with PCSK9 binding activity): LDLR-sECD-Fc (hIgG 1) as detection reagent

Annotation: the cross-hatched portion is a signal peptide, the double-hatched portion is a shortened version of the LDLR ectodomain fragment (LDLR-zecd) having binding activity to PCSK9, the italic portion is an hig 1-Fc portion;

a more shortened version of the LDLR ectodomain fragment was fused to the hIgG1-Fc fusion protein (with pCSK9 binding activity): LDLR-ssECD-Fc (hIgG 1) as detection reagent

Annotation: the cross-hatched portion is the signal peptide, the double-hatched portion is a more shortened version of the LDLR ectodomain fragment (LDLR-ssECD) with PCSK9 binding activity, and the italic portion is the hig 1-Fc portion.

EXAMPLE 2 purification of recombinant proteins related to PCSK9 and LDLR, purification of hybridoma antibodies and recombinant antibodies

1. Purification step of His-tagged recombinant protein:

isolating the cell expression supernatant at high speedThe heart was freed of impurities and the buffer was replaced by PBS and imidazole was added to a final concentration of 5mM. The nickel column was equilibrated with PBS containing 5mM imidazole and washed 2-5 column volumes. The displaced supernatant samples were subjected to IMAC column. The column was washed with PBS containing 5mM imidazole to A ₂₈₀ The reading drops to baseline. The column was then rinsed with PBS+10mM imidazole to remove non-specifically bound heteroproteins and the effluent was collected. The target protein was eluted with a PBS solution containing 300mM imidazole, and the elution peaks were collected. The collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE), the mobile phase being PBS. Removing the peak of the polymer, and collecting the eluting peak. The obtained protein is split into separate parts for standby after electrophoresis and peptide drawing, LC-MS identification are right. Resulting in His-tagged PCSK9-His6 (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2), PCSK9-TEV-His6 (SEQ ID NO: 3) PCSK9-D374Y-His6 (SEQ ID NO: 4), PCSK9-BP15-His6 (SEQ ID NO: 5), PCSK9-D374Y-BP15-His6 (SEQ ID NO: 6) for immunogens or detection reagents of the antibodies of the invention. Wherein PCSK9-TEV-His6 is purified and then subjected to enzyme digestion by TEV enzyme, and the enzyme digestion product is combined by an IMAC column to remove TEV enzyme, incompletely digested PCSK9-TEV-His6 or excised C-terminal domain fragment with His tag, and the PCSK9 fragment (abbreviated as N-PCSK 9) with only N-terminal domain is obtained by concentration in IMAC effluent liquid and used as immunogen for immunization of mice.

2. Purification step of His-tagged and Flag-tagged LDLR-ECD-Flag-His6 (SEQ ID NO: 7) recombinant protein:

The sample was centrifuged at high speed to remove impurities and concentrated to the appropriate volume. The flag affinity column was equilibrated with 0.5 XPBS and washed 2-5 column volumes. And loading the cell expression supernatant sample after impurity removal on a column. The column was washed with 0.5 XPBS to A ₂₈₀ The reading drops to baseline. The column was washed with PBS containing 0.3M NaCl, and the heteroproteins were washed and collected. The target protein was eluted with 0.1M acetic acid (pH 3.5-4.0), collected and pH adjusted to neutral. The collected eluate was concentrated and further purified by gel chromatography Superdex200 (GE), the mobile phase being PBS. Removing polymer peak, collecting eluting peak, collecting sample, electrophoresis, peptide mapping, LC-MS identification, and packaging. Obtaining the FLAG/His 6-tagged LDLR-ECD-FLAG-His6 (SEQ ID NO: 7) for the sex of the antibodies of the inventionCan be tested.

3. Purification step of Fc fusion protein of LDLR:

and (3) centrifuging the cell expression supernatant sample at a high speed to remove impurities, concentrating to a proper volume, and loading the concentrated cell expression supernatant sample on a Protein A column. The column was washed with PBS until the a280 reading dropped to baseline. The target protein was eluted with 100mM sodium acetate pH3.0 and neutralized with 1M Tris-HCl (Tris-hydroxymethyl aminomethane-HCl). The eluted sample is properly concentrated and then is further purified by gel chromatography Superdex200 (GE) balanced by PBS, and the peak of the polymer is collected and split charging is carried out for standby. This method was used to purify LDLR-sECD-Fc (hIgG 1) (SEQ ID NO: 8) and LDLR-ssECD-Fc (hIgG 1) (SEQ ID NO: 9). Both can be used as PCSK9 antibody functionality tests.

Example 3 preparation of anti-human PCSK9 hybridoma monoclonal antibodies

1. Immunization

Anti-human PCSK9 monoclonal antibodies are generated by immunizing mice. The experimental SJL white mice, females, 6 weeks old (Beijing Vitolihua laboratory animal technologies Co., ltd., animal production license number: SCXK 2012-0001). Feeding environment: SPF stage. After the mice are purchased, the mice are fed in a laboratory environment for 1 week, the light/dark period is regulated for 12/12 hours, and the temperature is 20-25 ℃; humidity is 40-60%. The acclimatized mice were immunized (A/B) in two protocols, 6-10 per group. The immune antigen is human PCSK9-His6 (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2) and N-PCSK9 (SEQ ID NO: 3) with His tag.

Scheme A was emulsified with Freund's adjuvant (sigma Lot Num: F5881/F5506): first Freund's complete adjuvant (CFA) was used, and the remainder was used to boost Freund's incomplete adjuvant (IFA). The ratio of antigen to adjuvant was 1:1, 100 μg/min (priming), 50 μg/min (boost). 100 μg/dose of emulsified antigen was injected Intraperitoneally (IP) on day 0, once every two weeks after priming for 6-8 weeks.

Scheme B was cross-immunized with Titermax (sigma Lot Num: T2684) and Alum (Threemo Lot Num: 77161). The ratio of antigen to adjuvant (titrimax) was 1:1, the ratio of antigen to adjuvant (Alum) was 3:1, 10-20 μg/per (priming), 5 μg/per (boosting). Day 0 Intraperitoneal (IP) injection of 20/10. Mu.g/dose of emulsified antigen was administered weekly after priming, alternating with Titermax and Alum for 6-11 weeks. Four weeks after immunization, back or intraperitoneal injections of antigen were selected based on back caking and abdominal swelling.

2. Cell fusion

Mice with high serum antibody titers (see test examples 1 and 2, below, ELISA methods for PCSK9 binding) and titers towards the plateau were selected for spleen cell fusion, and selected mice were immunized by sprinting 72 hours prior to fusion, PCSK9-His610 μg/mouse, i.p. Spleen lymphocytes and myeloma Sp2/0 cells are fused by adopting an optimized PEG-mediated fusion stepCRL-8287 ^TM ) And (5) fusing to obtain the hybridoma cells. The fused hybridoma cells were resuspended in HAT complete medium (RPMI-1640 medium containing 20% FBS, 1 XHAT and 1 XOPI) and plated in 96-well cell culture plates (1X 10) ⁵ 150 μl/well), 37℃and 5% CO ₂ And (5) incubating. HAT complete medium, 50. Mu.l/well, 37℃and 5% CO were added on day 5 after fusion ₂ And (5) incubating. From day 7 to day 8 after fusion, according to cell growth density, the whole culture medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1×HT and 1×OPI), 200 μl/well, 37℃and 5% CO ₂ And (5) incubating.

3. Hybridoma cell screening

ELISA assays for binding to PCSK9 or PCSK9-Y were performed on days 10-11 post-fusion, depending on cell growth density (see test examples 1 and 2). And performing blocking ELISA detection (see test examples 3 and 4) of PCSK9 or PCSK9-Y binding to LDLR on positive well cells combined with ELISA detection, replacing liquid from the positive wells, and timely expanding the positive wells into 24-well plates according to cell density. The cell lines engrafted into 24-well plates were retested for seed protection and first subcloning. Seed protection was performed with the first subclone selection (see test examples 1 and 2) positive and the second subclone was performed. The second subcloning was positive (see test examples 1 and 2) for seed protection and protein expression. Multiple fusions resulted in hybridoma cells that blocked PCSK9 or PCSK9-Y binding to LDLR (see test examples 3 and 4).

Hybridoma clone mAb-001 was obtained by blocking and binding assays, and antibodies were further prepared by serum-free cell culture and purified according to the purification examples for use in the test cases.

The murine anti-variable region sequence of hybridoma clone mAb-001 was determined as follows:

>mAb-001 VH

>mAb-001VL

note that: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italics in sequence is FR sequence and underlined is CDR sequence.

TABLE 1 heavy and light chain CDR region sequences of PCSK-9 antibody mAb-001

Example 4 humanization of anti-human PCSK9 hybridoma monoclonal antibodies

1. Hybridoma clone mAb-001 humanized framework selection

By comparing the germline gene database of the heavy and light chain variable region of the IMGT human antibody with MOE software, the germline genes of the heavy and light chain variable region with high homology with mAb-001 are respectively selected as templates, and the CDRs of the two murine antibodies are respectively transplanted into corresponding human templates to form variable region sequences with the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Wherein the amino acid residues are determined and annotated by the Kabat numbering system.

Humanized light chain templates of the murine antibody mAb-001 are IGKV1-39 x 01 and hjk2.1, humanized heavy chain templates are IGHV1-2 x 02 and hjh2, and the variable region sequences of the humanized antibody h001-1 obtained after humanization are as follows:

>h001-1 VH

>h001-1 VL

And (3) injection: the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, italics in sequence is FR sequence and underlined is CDR sequence.

2. Template selection and back-mutation design for hybridoma clone mAb-001, see table 2 below; the humanized sequence combinations after the back mutation of the hybridoma clones are shown in Table 4.

TABLE 2 design of hybridoma clone back mutations

Note that: S66D was mutated back to D according to the Kabat numbering system.

Grafted represents murine antibody CDR-implanted human germline FR region sequences, specific sequences for each mutant variable region are shown in table 3 below:

TABLE 3 variable region sequences of the mutants

Note that: the cross-line portion of the sequence is the CDR region.

TABLE 4 murine anti-mAb-001 humanized sequence combinations

	h001_VL.1	h001_VL.1A	h001_VL.1B	h001_VL.1C
					h001_VH.1	h001-1	h001-2	h001-3	h001-4
h001_VH.1A	h001-5	h001-6	h001-7	h001-8
					h001_VH.1B	h001-9	h001-10	h001-11	h001-12
h001_VH.1C	h001-13	h001-14	h001-15	h001-16
					h001_VH.1D	h001-17	h001-18	h001-19	h001-20
h001_VH.1E	h001-21	h001-22	h001-23	h001-24

Note that: the table shows the combinations of variable region portions of the humanized antibodies obtained by combining the various sequences and their mutated sequences. The variable region of the humanized antibody h001-1 consists of the light chain h001_VL1, the heavy chain h001_VH.1A, as represented by h 001-1. And so on.

3. The above humanized sequences were combined and subjected to antibody formation, the heavy chain constant region was derived from human IgG1, and the light chain constant region was derived from human kappa chain. Obtaining corresponding humanized antibody, and verifying that the obtained PCSK9 antibody has higher binding activity with PCSK9 and PCSK 9-Y; and can effectively block the combination between PCSK9/PCSK9-Y and LDLR.

EXAMPLE 5 construction and expression of humanized antibody IgG1 and IgG1-YTE form of anti-human PCSK9

The method for constructing and expressing the humanized antibody of the anti-human PCSK9 comprises the following steps:

1. primer design: the VH/VK gene fragment containing recombination is synthesized by designing a plurality of primers by using online software DNAWorks (v3.2.2) (works /). 5'-30bp Signal peptide+VH/VK+30bp CH1/CL-3'. Primer design principle: the target gene 2 is different from the target gene 1 in that the target gene is provided with 2 aa, and a primer with a mutation site is additionally arranged.

2. And (3) segment splicing: the gene fragment required for VH/VK recombination was obtained by two-step PCR amplification using the above designed primers according to TaKaRa Primer STAR GXL DNA polymerase protocol.

3. Construction of expression vector pHr (with Signal peptide and constant region Gene (CH 1-FC/CL) fragment) and cleavage

The expression vector pHr (with signal peptide and constant region gene (CH 1-FC/CL) fragment) was constructed using specific restriction endonucleases, such as BsmBI, with recognition sequences that differ from the cleavage site. BsmBI enzyme cutting carrier, cutting glue and recovering for standby.

4. Recombinant construction of expression vector VH-CH1-FC-pHr/VK-CL-pHr

The VH/VK gene fragment and BsmBI enzyme digestion recovery expression vector pHr (with signal peptide and constant region gene (CH 1-FC/CL) fragment) are respectively added into DH5 alpha competent cells according to the proportion of 3:1, ice bath is carried out at 0 ℃ for 30min, heat shock is carried out at 42 ℃ for 90 seconds, LB medium with 5 times volume is added, incubation is carried out at 37 ℃ for 45min, LB-Amp plates are coated, culture is carried out at 37 ℃ for overnight, and monoclonal sequencing is selected to obtain each target clone.

Antibodies of the invention may be, but are not limited to, the above design constructs. Taking h001-4 as an example, the design of antibodies and mutants thereof gave (1) h001-4-WT: the IgG1 version of h001-4, i.e., humanized sequences combine h001-4, with the heavy chain constant region from a human IgG1, with the light chain constant region from a human kappa chain; (2) h001-4-YTE: h001-4-IgG1-YTE form, i.e., humanized sequences combine h001-4, with the heavy chain constant region of a mutant human IgG1 (YTE mutation), with the light chain constant region from a human kappa chain. The mutated human IgG1 may also be another form of mutation.

Construction and expression of anti-human PCSK9 humanized antibodies (IgG 1 and IgG1-YTE versions of h 001-4) the sequences were as follows:

h001-4 IgG1 form, heavy chain constant region from human IgG1, light chain constant region from human kappa light chain:

heavy chain amino acid sequence (human IgG 1):

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGYINPSSGFTKYHQNFKDRVTMTRDTSISTAYMELSRLRSDDTAVYYCARQYDYDEDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：28

heavy chain DNA sequence:

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCCAGTGCCAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCCGGAGCGAGCGTAAAGGTGAGCTGCAAGGCCAGCGGATACACCTTCACCGACTACTGGATGCACTGGGTGAGGCAGGCCCCAGGACAGGGCCTGGAGTGGATGGGCTACATCAACCCCAGCAGCGGCTTTACCAAGTATCACCAGAACTTCAAAGACAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCAGGCTGAGGAGCGACGACACCGCCGTGTACTACTGCGCCAGGCAATACGACTACGACGAGGACTGGTACTTCGACGTGTGGGGCCAAGGAACCACCGTGACTGTGAGCAGCGCTTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO：29

h001-4-kappa

light chain amino acid sequence:

DIVMSQSPSSLSASVGDRVTITCKSSQSLLNSRTRKNFLAWYQQKPGKSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCKQSFNLFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO：30

light chain DNA sequence:

ATGGACATGCGCGTGCCCGCCCAGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGCGACATCGTGATGTCTCAGAGCCCATCTAGCCTGAGCGCCAGCGTGGGCGACAGGGTAACCATCACCTGCAAGAGCAGCCAAAGCCTGCTGAACAGCAGGACCCGCAAGAACTTCCTGGCTTGGTATCAGCAGAAGCCCGGCAAGTCTCCCAAGTTGCTGATCTACTGGGCCAGCACCAGGGAGAGCGGCGTGCCCGACAGGTTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTGACCATCTCTAGTCTGCAGCCCGAGGACTTCGCCACCTACTACTGCAAGCAGAGCTTCAATCTGTTCACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

SEQ ID NO：31

the h001-4-YTE light chain is h001-4-kappa: SEQ ID NO:30

The h001-4-YTE heavy chain amino acid sequence is as follows:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWMHWVRQAPGQGLEWMGYINPSSGFTKYHQNFKDRVTMTRDTSISTAYMELSRLRSDDTAVYYCARQYDYDEDWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ ID NO：32

h001-4-YTE heavy chain DNA sequence:

ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCCAGTGCCAGGTGCAGCTGGTGCAGAGCGGCGCTGAGGTGAAGAAGCCCGGAGCGAGCGTAAAGGTGAGCTGCAAGGCCAGCGGATACACCTTCACCGACTACTGGATGCACTGGGTGAGGCAGGCCCCAGGACAGGGCCTGGAGTGGATGGGCTACATCAACCCCAGCAGCGGCTTTACCAAGTATCACCAGAACTTCAAAGACAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAGCTGAGCAGGCTGAGGAGCGACGACACCGCCGTGTACTACTGCGCCAGGCAATACGACTACGACGAGGACTGGTACTTCGACGTGTGGGGCCAAGGAACCACCGTGACTGTGAGCAGCGCTTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCTACATCACCCGGGAGCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

SEQ ID NO：33

remarks: underlined is the signal peptide DNA sequence

Exemplary antibody pharmaceutical composition (formulation) preparation Process

The first step: a quantity of purified PCSK-9 antibody (e.g., h 001-4-YTE) stock solution was taken, subjected to solvent displacement (preferably ultrafiltration) with an antibody-free buffer (e.g., 10mM, pH 6.0 histidine-HCl buffer), and the protein was concentrated to 60mg/mL (+ -2 mg/mL) by 6-fold volume displacement through an ultrafiltration membrane. Adding a certain volume of sucrose mother liquor, and uniformly mixing to obtain the final sucrose concentration of 25mg/mL. Adding a certain volume of Tween-80 mother solution, and mixing to obtain final Tween-80 concentration of 0.2mg/mL. 10mM histidine buffer pH 6.0 was added to volume to give a protein concentration of 50mg/mL (+ -5 mg/mL) and the final product pH was about 6.3+ -0.1 (other formulations to be tested or stability formulations were formulated with reference to similar procedures).

The product is filtered, sampled and detected asepsis by central control. The stock solution was filtered through a 0.22 μm PVDF filter cartridge and the filtrate was collected.

And a second step of: the filling amount is regulated to 3.6ml, the filtrate is filled in a 6ml penicillin bottle, and the filling amount difference is detected by controlling the sampling center at the beginning, the middle and the end of filling respectively.

And a third step of: and filling the filled and plugged liquid medicine into a freeze-drying box, and freeze-drying. The lyophilization procedure is prefreezing, primary drying and secondary drying. After the lyophilization procedure was completed, vacuum stoppered.

The time used for freeze-drying can be adjusted along with actual conditions, and the model of a freeze dryer, the loading capacity of freeze-dried medicaments and the containers of the freeze-dried medicaments can all influence the freeze-drying time. Such time adjustments are well known to those skilled in the art.

Fourth step: and opening the capping machine, adding an aluminum cap, and capping.

Fifth step: and (5) visual inspection, and confirming that the product has no defects of collapse, inaccurate loading and the like. Printing and pasting a penicillin bottle label; printing paper box labels, folding paper boxes, boxing and sticking paper box labels.

Example 6 screening of buffer System

An anti-PCSK-9 antibody h001-4-YTE preparation was formulated at a protein concentration of 1mg/ml with the following buffers:

1) 20mM citric acid-sodium citrate, pH 4.0

2) 20mM citric acid-sodium citrate, pH 4.5

3) 20mM citric acid-sodium citrate, pH 5.0

4) 20mM citric acid-sodium citrate, pH 5.5

5) 20mM citric acid-sodium citrate, pH6.0

6) 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH6.0

7) 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.5

8) 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.0

9) 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 7.5

10 20mM tris (hydroxymethyl) aminomethane-HCl, pH 7.5

11 20mM tris-hydroxymethyl-aminomethane-HCl, pH 8.0

12 20mM tris-hydroxymethyl-aminomethane-HCl, pH 8.5

The thermal stability of the anti-PCSK-9 antibodies in each formulation was measured by differential scanning calorimetry (differential scanning calorimetry, DSC). Thermal denaturation midpoint temperature (Tm) analysis of the medicine shows that the anti-PCSK-9 antibody has better thermal stability when the pH is more than or equal to 6.0. The results are shown in Table 5. Buffers between pH6.0 and 6.5 were therefore selected: histidine-hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, succinic acid-sodium succinate, citric acid-sodium citrate, for subsequent study.

TABLE 5H001-4-YTE buffer-pH DSC screening results

Note that: N/A indicates that the component was not added.

An anti-PCSK-9 antibody preparation was formulated at a protein concentration of 1mg/ml with the following buffers:

1) 20mM histidine-hydrochloride, pH6.0

2) 20mM histidine-hydrochloride, pH 6.5

3) 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.0

4) 20mM sodium dihydrogen phosphate-disodium hydrogen phosphate, pH 6.5

5) 20mM sodium succinate, pH 6.0

6) 20mM citric acid-sodium citrate, pH 6.0

7) 20mM citric acid-sodium citrate, pH 6.5

The thermal stability of the anti-PCSK-9 antibodies in each formulation was measured by differential scanning calorimetry (differential scanning calorimetry, DSC). Thermal denaturation midpoint temperature (Tm) analysis of the drug product showed (see table 6) that the stability of the anti-PCSK-9 antibodies in histidine, phosphate and succinate buffer systems was slightly better than the citrate buffer system.

TABLE 6H001-4-YTE buffer-pH DSC screening results

/>

Note that: N/A indicates that the component was not added.

Example 7 screening of sugar in formulations

anti-PCSK-9 antibody preparations with a PCSK-9 antibody protein concentration of 150mg/ml were prepared with buffers of the following different sugar classes:

1) 20mM histidine-hydrochloride, 70mg/ml sucrose, pH 6.5

2) 20mM histidine-hydrochloride, 70mg/ml alpha, alpha-trehalose dihydrate, pH 6.5

Each formulation was filtered and filled into 2mL neutral borosilicate glass tube injection vials at 0.5 mL/vial, with the water needle tamponade capped. The samples were taken and subjected to high temperature and low temperature illumination at 40℃and freeze thawing cycle (the samples were left at-20℃for 24 hours and then left at room temperature to completely melt and mix the samples into one cycle), and the results showed that the stability of sucrose and trehalose against PCSK-9 antibody was similar, and sucrose was selected as the stabilizer for the anti-PCSK-9 antibody, and the results are shown in Table 7.

TABLE 7 accelerated test results of different sugar types of H001-4-YTE

Antibody preparations with PCSK-9 antibody protein concentrations of 150mg/ml were prepared with the following buffers of different sucrose concentrations:

1) 20mM histidine-hydrochloride, 0mg/ml sucrose, pH 6.5

2) 20mM histidine-hydrochloride, 10mg/ml sucrose, pH 6.5

3) 20mM histidine-hydrochloride, 40mg/ml sucrose, pH 6.5

4) 20mM histidine-hydrochloride, 70mg/ml sucrose, pH 6.5

The osmotic pressure value shows that when the sucrose is more than or equal to 70mg/ml, the osmotic pressure meets the minimum subcutaneous injection requirement. The PCSK-9 antibody preparation is prepared by adopting a dilute freeze-dissolution process (three-volume stock solution is freeze-dried, one-volume injection is re-dissolved by water), and the invention is not particularly described to adopt the ratio freeze-dissolution process, so that the osmotic pressure requirement of injection is met when the sugar concentration in a final prescription (re-dissolved preparation) is 75mg/ml, the sugar concentration in the stock solution is conveniently set to 25mg/ml, and meanwhile, the sugar concentration of 25mg/ml is easy to freeze-dry. Thus, the sugar concentration in the final formulation was selected to be 75mg/ml, and the sugar concentration in the stock solution was determined to be 25mg/ml.

TABLE 8 determination of osmolarity of H001-4 YTE sugar concentration

Note that: N/A indicates that the component was not added.

Example 8 screening of surfactants in formulations

anti-PCSK-9 antibody formulations with PCSK-9 antibody protein concentrations of 150mg/ml were prepared with buffers of different concentrations of surfactants:

1) 20mM histidine-hydrochloride, pH 6.5

2) 20mM histidine-hydrochloride, 0.2mg/ml polysorbate 20, pH 6.5

3) 20mM histidine-hydrochloride, 0.4mg/ml polysorbate 80, pH 6.5

Each formulation was filtered and filled into 2mL neutral borosilicate glass tube injection vials at 0.5 mL/vial, with a water needle stopper tamponade capped. The sample was placed on a constant temperature shaking table at 25℃and shaken at 300 rpm. The appearance results show that the surfactant effectively prevents aggregation of the anti-PCSK-9 antibody. While polysorbate 20 and polysorbate 80 have no significant difference in effect on H001-4-YTE, we selected polysorbate 80 as the stabilizer for anti-PCSK-9 antibodies.

TABLE 9 Effect of surfactants on H001-4-YTE aggregation at 25 ℃,300rpm shaking

Example 9 screening and confirmation of buffer System in formulations

Formulations containing 25mg/ml sucrose, 0.2mg/ml polysorbate 80 and 50mg/ml antibody protein were prepared with buffers containing 20mM histidine-hydrochloride or 20mM sodium succinate at pH6.0 or 6.5. Each formulation was filtered and lyophilized by filling 3.6 mL/vial into 6mL neutral borosilicate glass tube injection vials sealed with a freeze-dried sterile powder with a halobutyl rubber stopper. Storing the lyophilized product at 2-8deg.C, 25deg.C and 40deg.C, and redissolving with injectable water for stability analysis. The results show that anti-PCSK-9 antibodies are very stable at both 2-8 ℃ and 25 ℃. However, the reconstituted appearance on day 0 showed a clear opalescence of the pH6.0 succinic buffer compared to the His system (not shown in the table), and thus the histidine system was chosen as the buffer system for the anti-PCSK-9 antibody.

TABLE 10 stability of H001-4-YTE freeze-dried powder at different temperatures

Example 10 comprehensive screening of formulation Components

The anti-PCSK-9 antibody preparation containing 25mg/ml sucrose was prepared with 50mg/ml PCSK-9 antibody protein concentration in buffers containing the following surfactants at different concentrations, different pH's and different ionic strengths:

1) 10mM histidine-hydrochloride, 0.1mg/ml polysorbate 80, pH 6.5

2) 10mM histidine-hydrochloride, 0.3mg/ml polysorbate 80, pH 6.0

3) 10mM histidine-hydrochloride, 0.2mg/ml polysorbate 80, pH 5.5

4) 15mM histidine-hydrochloride, 0.3mg/ml polysorbate 80, pH 5.5

5) 10mM histidine-hydrochloride, 0.2mg/ml polysorbate 80, pH 6.0

6) 15mM histidine-hydrochloride, 0.3mg/ml polysorbate 80, pH 6.5

7) 15mM histidine-hydrochloride, 0.2mg/ml polysorbate 80, pH 6.0

8) 5mM histidine-hydrochloride, 0.3mg/ml polysorbate 80, pH 5.5

9) 15mM histidine-hydrochloride, 0.1mg/ml polysorbate 80, pH 5.5

10 5mM histidine-hydrochloride, 0.1mg/ml polysorbate 80, pH 6.0

11 5mM histidine-hydrochloride, 0.2mg/ml polysorbate 80, pH 6.5

Each formulation was filtered and lyophilized by filling 3.6 mL/vial into 6mL neutral borosilicate glass tube injection vials sealed with a freeze-dried sterile powder with a halobutyl rubber stopper. The samples were stored at 40℃for stability analysis and data analysis were performed as the difference in SEC monomers, which showed that the preparation of PCSK-9 antibody using polysorbate 80 at an ionic strength of pH 6.0, 10mM histidine-HCl buffer, and 0.2mg/ml was more favorable for the stability of PCSK-9 antibody at a protein concentration of 50mg/ml and 25mg/ml sucrose. After lyophilization and reconstitution, the final formulation had 3 times the concentration of each component (150 mg/ml protein, 75mg/ml sucrose, 30mM histidine-HCl buffer, 0.6mg/ml polysorbate 80) as compared to the pre-lyophilization stock solution, and a pH of 6.3.+ -. 0.1.

TABLE 11 results of high temperature experiments with H001-4-YTE

Example 11 optimization of Primary drying temperature

An anti-PCSK-9 antibody preparation having a PCSK-9 antibody protein concentration of 50mg/ml, 25mg/ml sucrose, 0.2mg/ml polysorbate 80 was prepared with a buffer containing 10mM histidine-hydrochloride at pH 6.0. The antibodies were filled into 6mL penicillin bottles at 3.6 mL/bottle, lyophilized at-14 ℃ and-5 ℃ primary drying temperature, respectively, and sealed with a lyophilized rubber stopper. The reconstituted samples were subjected to lyophilization before and after comparison. The results show that-5 ℃ is the primary drying temperature with better freeze-drying process.

TABLE 12 comparison of samples prepared by different drying techniques for H001-4-YTE before and after lyophilization

EXAMPLE 12 determination of compatibility of formulations with different Material containers

H001-4-YTE was formulated at 50mg/ml in pH 6.0, 10mM histidine-hydrochloride, 25mg/ml sucrose, 0.2mg/ml polysorbate 80. The formulations were filled into glass bottles, liquid storage bags and 316L stainless steel jars, respectively, and left at 2-8deg.C for 24 hours. Analysis of protein content and purity showed that H001-4-YTE was stable for 24 hours. The formulation was compatible with 316L stainless steel cans, glass vials and liquid storage bags.

TABLE 13 stability of H001-4-YTE in different contact materials

Example 13 determination of compatibility of formulations with different Filter membranes

H001-4-YTE was formulated at 50mg/ml in 10mM histidine-hydrochloride buffer, pH 6.0, 25mg/ml sucrose, 0.2mg/ml polysorbate 80. The preparation was filtered through 0.22 μm PES and PVDF filters and sampled and tested at 30min and 1h, respectively. Analysis of protein content, appearance and purity showed that H001-4-YTE was stable for 1 hour in contact with the filter. The formulation is compatible with both PES and PVDF filters.

TABLE 14 stability of H001-4-YTE in different filter materials

Example 14, other alternative formulation formulations

The pharmaceutical composition of the invention can be used as a stock solution of a pharmaceutical preparation or directly used as an injection. When the pharmaceutical composition is used as a stock solution of a pharmaceutical preparation, the pharmaceutical composition is prepared into a freeze-dried preparation through a freeze-drying process, and the freeze-dried preparation is re-dissolved to form an injection for clinical use. The invention prepares the freeze-dried preparation according to the dilute freeze-drying and concentrated dissolving process, i.e. the stock solution of the pharmaceutical preparation with low component concentration is freeze-dried to obtain the freeze-dried preparation, and the freeze-dried preparation is re-dissolved into the pharmaceutical composition with higher concentration for clinical use when in use. The storage stability of the lyophilized formulation is longer than that of the liquid formulation. The higher the concentration of each component, the longer the lyophilization time of the stock pharmaceutical preparation used for the lyophilized preparation. The concentration of each component of the original medicinal preparation is 2-5 times of that of the reconstituted injection according to the index setting of the comprehensive freeze-drying process, and the preferred embodiment of the invention is 3 times.

The present invention provides a stable pharmaceutical formulation comprising: combination of PCSK-9 antibody protein (non-limiting example h 001-4-YTE) and optionally a stabilizing buffer from the following:

(1) Antibody h001-4-YTE 150mg/ml,75mg/ml sucrose, polysorbate 80 0.6mg/ml, and 30mM histidine-hydrochloride buffer, final pH 6.4;

(2) Antibody h001-4-YTE 150mg/ml,75mg/ml sucrose, polysorbate 80 0.6mg/ml, and 30mM histidine-hydrochloride buffer, final pH 6.2;

(3) Antibody h001-4-YTE 150mg/ml,75mg/ml sucrose, polysorbate 80 0.6mg/ml, and 30mM histidine-hydrochloride buffer, final pH 6.3;

(4) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 10mM histidine-hydrochloride buffer, final pH 6.4;

(5) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 10mM histidine-hydrochloride buffer, final pH 6.3;

(6) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 10mM histidine-hydrochloride buffer, final pH 6.2;

(7) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 10mM histidine-hydrochloride buffer, final pH 6.1;

(8) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 10mM histidine-hydrochloride buffer, final pH 6.0;

(9) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 10mM histidine-hydrochloride buffer, final pH 6.5;

(10) Antibody h001-4 50mg/ml,25mg/ml sucrose, 0.2mg/ml polysorbate 80, and 10mM histidine-hydrochloride buffer, final pH 6.3;

(11) Antibody h001-4 50mg/ml,25mg/ml sucrose, 0.2mg/ml polysorbate 80, and 10mM histidine-hydrochloride buffer, final pH 6.2;

(12) Antibody h001-4 50mg/ml,25mg/ml sucrose, 0.2mg/ml polysorbate 80, and 10mM histidine-hydrochloride buffer, final pH 6.4;

(13) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.1mg/ml, and 20mM histidine-hydrochloride buffer, final pH 6.3;

(14) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 15mM histidine-hydrochloride buffer, final pH 6.2;

(15) Antibody h001-4-YTE 50mg/ml,25mg/ml sucrose, polysorbate 80 at 0.2mg/ml, and 20mM histidine-HCl buffer, final pH 6.4.

(16) Antibody h001-4-YTE 30mg/ml,10mg/ml sucrose, polysorbate 80 0.05mg/ml, and 5mM histidine-hydrochloride buffer pH 5.5;

(17) Antibody h001-4-YTE 70mg/ml,75mg/ml sucrose, polysorbate 80 at 0.6mg/ml, and 30mM histidine-hydrochloride buffer at pH 6.5;

(18) Antibody h001-4-YTE 45mg/ml,20mg/ml sucrose, 0.1mg/ml polysorbate 80, and 8mM histidine-hydrochloride buffer pH 6.0;

(19) Antibody h001-4-YTE 55mg/ml, sucrose 40mg/ml, polysorbate 80 0.3mg/ml, and 15mM histidine-HCl buffer pH 6.2.

The pharmaceutical composition of the invention can be prepared into corresponding freeze-dried preparation by freeze-drying process, and the freeze-dried preparation can be re-dissolved by injection liquid medicine to obtain the following pharmaceutical composition for clinical use:

(1) Antibody PCSK-9 antibody protein 150mg/ml,75mg/ml sucrose, 0.6mg/ml polysorbate 80, and 20mM histidine-hydrochloride buffer, final pH 6.3.+ -. 0.1;

(2) Antibody PCSK-9 antibody protein 120mg/ml,55mg/ml sucrose, 0.4mg/ml polysorbate 80, and 10mM histidine-hydrochloride buffer, final pH 6.0;

(3) Antibody PCSK-9 antibody protein 200mg/ml,95mg/ml sucrose, 0.8mg/ml polysorbate 80, and 30mM histidine-hydrochloride buffer, final pH 6.5.

While particular embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these are merely illustrative, and that many changes and modifications may be made to these embodiments without departing from the principles and spirit of the invention. Accordingly, the scope of the invention is defined by the appended claims.

SEQUENCE LISTING

<110> Jiangsu Hengrui medicine Co., ltd

Shanghai Hengrui medicine Co., ltd

<120> a PCSK-9 antibody pharmaceutical composition and use thereof

<130> P19113708CP

<150> CN 201710519829.6

<151> 2017-06-30

<160> 33

<170> PatentIn version 3.5

<210> 1

<211> 698

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> His-tagged pCSK9-PADRE-His6 for use in immunogenic immunized mice or detection reagents

<400> 1

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln His His His His His His

690 695

<210> 2

<211> 714

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> pCSK9-PADRE-His6 with PADRE peptide and His tag as immunogen, PADRs contained

E-peptide can promote immunity

<400> 2

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln Gly Ser Gly Ala Lys Phe Val Ala Ala Trp Thr Leu

690 695 700

Lys Ala Ala Ala His His His His His His

705 710

<210> 3

<211> 704

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> with TEV cleavage site

pCSK9 and his tag fusion: pCSK9-TEV-His6 can be cut by TEV enzyme to obtain N-pCSK9 #

N-terminal pCSK9 domain) as an immunogen

<400> 3

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Glu Asn Leu Tyr Phe Gln Gly Ala Gly Trp Gln Leu Phe Cys Arg

450 455 460

Thr Val Trp Ser Ala His Ser Gly Pro Thr Arg Met Ala Thr Ala Val

465 470 475 480

Ala Arg Cys Ala Pro Asp Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser

485 490 495

Arg Ser Gly Lys Arg Arg Gly Glu Arg Met Glu Ala Gln Gly Gly Lys

500 505 510

Leu Val Cys Arg Ala His Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala

515 520 525

Ile Ala Arg Cys Cys Leu Leu Pro Gln Ala Asn Cys Ser Val His Thr

530 535 540

Ala Pro Pro Ala Glu Ala Ser Met Gly Thr Arg Val His Cys His Gln

545 550 555 560

Gln Gly His Val Leu Thr Gly Cys Ser Ser His Trp Glu Val Glu Asp

565 570 575

Leu Gly Thr His Lys Pro Pro Val Leu Arg Pro Arg Gly Gln Pro Asn

580 585 590

Gln Cys Val Gly His Arg Glu Ala Ser Ile His Ala Ser Cys Cys His

595 600 605

Ala Pro Gly Leu Glu Cys Lys Val Lys Glu His Gly Ile Pro Ala Pro

610 615 620

Gln Glu Gln Val Thr Val Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly

625 630 635 640

Cys Ser Ala Leu Pro Gly Thr Ser His Val Leu Gly Ala Tyr Ala Val

645 650 655

Asp Asn Thr Cys Val Val Arg Ser Arg Asp Val Ser Thr Thr Gly Ser

660 665 670

Thr Ser Glu Gly Ala Val Thr Ala Val Ala Ile Cys Cys Arg Ser Arg

675 680 685

His Leu Ala Gln Ala Ser Gln Glu Leu Gln His His His His His His

690 695 700

<210> 4

<211> 698

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> pCSK9-D374Y mutein, his-tagged: pCSK9-D374Y-His6 as detection reagent

<400> 4

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Tyr Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln His His His His His His

690 695

<210> 5

<211> 719

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> pCSK9 protein with biotin-accepting peptide inserted and his tag: pCSK9-BP15-His6 as a test

The agent, BP15 peptide position can carry out biotin labeling in the expression process, so that in vitro biotin labeling and in vitro biotin labeling are avoided

Construction variations that may result

<400> 5

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Asp Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln Gly Ser Thr Ser Gly Ser Gly Leu Asn Asp Ile Phe

690 695 700

Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His His

705 710 715

<210> 6

<211> 719

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> pCSK9 with biotin-receiving peptide and his tag inserted therein

D374Y mutant protein: pCSK9-D374Y-BP15-His6, detection protein

<400> 6

Met Gly Thr Val Ser Ser Arg Arg Ser Trp Trp Pro Leu Pro Leu Leu

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ala Arg Ala Gln Glu

20 25 30

Asp Glu Asp Gly Asp Tyr Glu Glu Leu Val Leu Ala Leu Arg Ser Glu

35 40 45

Glu Asp Gly Leu Ala Glu Ala Pro Glu His Gly Thr Thr Ala Thr Phe

50 55 60

His Arg Cys Ala Lys Asp Pro Trp Arg Leu Pro Gly Thr Tyr Val Val

65 70 75 80

Val Leu Lys Glu Glu Thr His Leu Ser Gln Ser Glu Arg Thr Ala Arg

85 90 95

Arg Leu Gln Ala Gln Ala Ala Arg Arg Gly Tyr Leu Thr Lys Ile Leu

100 105 110

His Val Phe His Gly Leu Leu Pro Gly Phe Leu Val Lys Met Ser Gly

115 120 125

Asp Leu Leu Glu Leu Ala Leu Lys Leu Pro His Val Asp Tyr Ile Glu

130 135 140

Glu Asp Ser Ser Val Phe Ala Gln Ser Ile Pro Trp Asn Leu Glu Arg

145 150 155 160

Ile Thr Pro Pro Arg Tyr Arg Ala Asp Glu Tyr Gln Pro Pro Asp Gly

165 170 175

Gly Ser Leu Val Glu Val Tyr Leu Leu Asp Thr Ser Ile Gln Ser Asp

180 185 190

His Arg Glu Ile Glu Gly Arg Val Met Val Thr Asp Phe Glu Asn Val

195 200 205

Pro Glu Glu Asp Gly Thr Arg Phe His Arg Gln Ala Ser Lys Cys Asp

210 215 220

Ser His Gly Thr His Leu Ala Gly Val Val Ser Gly Arg Asp Ala Gly

225 230 235 240

Val Ala Lys Gly Ala Ser Met Arg Ser Leu Arg Val Leu Asn Cys Gln

245 250 255

Gly Lys Gly Thr Val Ser Gly Thr Leu Ile Gly Leu Glu Phe Ile Arg

260 265 270

Lys Ser Gln Leu Val Gln Pro Val Gly Pro Leu Val Val Leu Leu Pro

275 280 285

Leu Ala Gly Gly Tyr Ser Arg Val Leu Asn Ala Ala Cys Gln Arg Leu

290 295 300

Ala Arg Ala Gly Val Val Leu Val Thr Ala Ala Gly Asn Phe Arg Asp

305 310 315 320

Asp Ala Cys Leu Tyr Ser Pro Ala Ser Ala Pro Glu Val Ile Thr Val

325 330 335

Gly Ala Thr Asn Ala Gln Asp Gln Pro Val Thr Leu Gly Thr Leu Gly

340 345 350

Thr Asn Phe Gly Arg Cys Val Asp Leu Phe Ala Pro Gly Glu Asp Ile

355 360 365

Ile Gly Ala Ser Ser Tyr Cys Ser Thr Cys Phe Val Ser Gln Ser Gly

370 375 380

Thr Ser Gln Ala Ala Ala His Val Ala Gly Ile Ala Ala Met Met Leu

385 390 395 400

Ser Ala Glu Pro Glu Leu Thr Leu Ala Glu Leu Arg Gln Arg Leu Ile

405 410 415

His Phe Ser Ala Lys Asp Val Ile Asn Glu Ala Trp Phe Pro Glu Asp

420 425 430

Gln Arg Val Leu Thr Pro Asn Leu Val Ala Ala Leu Pro Pro Ser Thr

435 440 445

His Gly Ala Gly Trp Gln Leu Phe Cys Arg Thr Val Trp Ser Ala His

450 455 460

Ser Gly Pro Thr Arg Met Ala Thr Ala Val Ala Arg Cys Ala Pro Asp

465 470 475 480

Glu Glu Leu Leu Ser Cys Ser Ser Phe Ser Arg Ser Gly Lys Arg Arg

485 490 495

Gly Glu Arg Met Glu Ala Gln Gly Gly Lys Leu Val Cys Arg Ala His

500 505 510

Asn Ala Phe Gly Gly Glu Gly Val Tyr Ala Ile Ala Arg Cys Cys Leu

515 520 525

Leu Pro Gln Ala Asn Cys Ser Val His Thr Ala Pro Pro Ala Glu Ala

530 535 540

Ser Met Gly Thr Arg Val His Cys His Gln Gln Gly His Val Leu Thr

545 550 555 560

Gly Cys Ser Ser His Trp Glu Val Glu Asp Leu Gly Thr His Lys Pro

565 570 575

Pro Val Leu Arg Pro Arg Gly Gln Pro Asn Gln Cys Val Gly His Arg

580 585 590

Glu Ala Ser Ile His Ala Ser Cys Cys His Ala Pro Gly Leu Glu Cys

595 600 605

Lys Val Lys Glu His Gly Ile Pro Ala Pro Gln Glu Gln Val Thr Val

610 615 620

Ala Cys Glu Glu Gly Trp Thr Leu Thr Gly Cys Ser Ala Leu Pro Gly

625 630 635 640

Thr Ser His Val Leu Gly Ala Tyr Ala Val Asp Asn Thr Cys Val Val

645 650 655

Arg Ser Arg Asp Val Ser Thr Thr Gly Ser Thr Ser Glu Gly Ala Val

660 665 670

Thr Ala Val Ala Ile Cys Cys Arg Ser Arg His Leu Ala Gln Ala Ser

675 680 685

Gln Glu Leu Gln Gly Ser Thr Ser Gly Ser Gly Leu Asn Asp Ile Phe

690 695 700

Glu Ala Gln Lys Ile Glu Trp His Glu His His His His His His

705 710 715

<210> 7

<211> 802

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> Flag-and His-tagged pCSK9 receptor protein LDLR ectodomain fragment: LDLR-ECD-Flag-H

is6, detection reagent

<400> 7

Met Gly Pro Trp Gly Trp Lys Leu Arg Trp Thr Val Ala Leu Leu Leu

1 5 10 15

Ala Ala Ala Gly Thr Ala Val Gly Asp Arg Cys Glu Arg Asn Glu Phe

20 25 30

Gln Cys Gln Asp Gly Lys Cys Ile Ser Tyr Lys Trp Val Cys Asp Gly

35 40 45

Ser Ala Glu Cys Gln Asp Gly Ser Asp Glu Ser Gln Glu Thr Cys Leu

50 55 60

Ser Val Thr Cys Lys Ser Gly Asp Phe Ser Cys Gly Gly Arg Val Asn

65 70 75 80

Arg Cys Ile Pro Gln Phe Trp Arg Cys Asp Gly Gln Val Asp Cys Asp

85 90 95

Asn Gly Ser Asp Glu Gln Gly Cys Pro Pro Lys Thr Cys Ser Gln Asp

100 105 110

Glu Phe Arg Cys His Asp Gly Lys Cys Ile Ser Arg Gln Phe Val Cys

115 120 125

Asp Ser Asp Arg Asp Cys Leu Asp Gly Ser Asp Glu Ala Ser Cys Pro

130 135 140

Val Leu Thr Cys Gly Pro Ala Ser Phe Gln Cys Asn Ser Ser Thr Cys

145 150 155 160

Ile Pro Gln Leu Trp Ala Cys Asp Asn Asp Pro Asp Cys Glu Asp Gly

165 170 175

Ser Asp Glu Trp Pro Gln Arg Cys Arg Gly Leu Tyr Val Phe Gln Gly

180 185 190

Asp Ser Ser Pro Cys Ser Ala Phe Glu Phe His Cys Leu Ser Gly Glu

195 200 205

Cys Ile His Ser Ser Trp Arg Cys Asp Gly Gly Pro Asp Cys Lys Asp

210 215 220

Lys Ser Asp Glu Glu Asn Cys Ala Val Ala Thr Cys Arg Pro Asp Glu

225 230 235 240

Phe Gln Cys Ser Asp Gly Asn Cys Ile His Gly Ser Arg Gln Cys Asp

245 250 255

Arg Glu Tyr Asp Cys Lys Asp Met Ser Asp Glu Val Gly Cys Val Asn

260 265 270

Val Thr Leu Cys Glu Gly Pro Asn Lys Phe Lys Cys His Ser Gly Glu

275 280 285

Cys Ile Thr Leu Asp Lys Val Cys Asn Met Ala Arg Asp Cys Arg Asp

290 295 300

Trp Ser Asp Glu Pro Ile Lys Glu Cys Gly Thr Asn Glu Cys Leu Asp

305 310 315 320

Asn Asn Gly Gly Cys Ser His Val Cys Asn Asp Leu Lys Ile Gly Tyr

325 330 335

Glu Cys Leu Cys Pro Asp Gly Phe Gln Leu Val Ala Gln Arg Arg Cys

340 345 350

Glu Asp Ile Asp Glu Cys Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys

355 360 365

Val Asn Leu Glu Gly Gly Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln

370 375 380

Leu Asp Pro His Thr Lys Ala Cys Lys Ala Val Gly Ser Ile Ala Tyr

385 390 395 400

Leu Phe Phe Thr Asn Arg His Glu Val Arg Lys Met Thr Leu Asp Arg

405 410 415

Ser Glu Tyr Thr Ser Leu Ile Pro Asn Leu Arg Asn Val Val Ala Leu

420 425 430

Asp Thr Glu Val Ala Ser Asn Arg Ile Tyr Trp Ser Asp Leu Ser Gln

435 440 445

Arg Met Ile Cys Ser Thr Gln Leu Asp Arg Ala His Gly Val Ser Ser

450 455 460

Tyr Asp Thr Val Ile Ser Arg Asp Ile Gln Ala Pro Asp Gly Leu Ala

465 470 475 480

Val Asp Trp Ile His Ser Asn Ile Tyr Trp Thr Asp Ser Val Leu Gly

485 490 495

Thr Val Ser Val Ala Asp Thr Lys Gly Val Lys Arg Lys Thr Leu Phe

500 505 510

Arg Glu Asn Gly Ser Lys Pro Arg Ala Ile Val Val Asp Pro Val His

515 520 525

Gly Phe Met Tyr Trp Thr Asp Trp Gly Thr Pro Ala Lys Ile Lys Lys

530 535 540

Gly Gly Leu Asn Gly Val Asp Ile Tyr Ser Leu Val Thr Glu Asn Ile

545 550 555 560

Gln Trp Pro Asn Gly Ile Thr Leu Asp Leu Leu Ser Gly Arg Leu Tyr

565 570 575

Trp Val Asp Ser Lys Leu His Ser Ile Ser Ser Ile Asp Val Asn Gly

580 585 590

Gly Asn Arg Lys Thr Ile Leu Glu Asp Glu Lys Arg Leu Ala His Pro

595 600 605

Phe Ser Leu Ala Val Phe Glu Asp Lys Val Phe Trp Thr Asp Ile Ile

610 615 620

Asn Glu Ala Ile Phe Ser Ala Asn Arg Leu Thr Gly Ser Asp Val Asn

625 630 635 640

Leu Leu Ala Glu Asn Leu Leu Ser Pro Glu Asp Met Val Leu Phe His

645 650 655

Asn Leu Thr Gln Pro Arg Gly Val Asn Trp Cys Glu Arg Thr Thr Leu

660 665 670

Ser Asn Gly Gly Cys Gln Tyr Leu Cys Leu Pro Ala Pro Gln Ile Asn

675 680 685

Pro His Ser Pro Lys Phe Thr Cys Ala Cys Pro Asp Gly Met Leu Leu

690 695 700

Ala Arg Asp Met Arg Ser Cys Leu Thr Glu Ala Glu Ala Ala Val Ala

705 710 715 720

Thr Gln Glu Thr Ser Thr Val Arg Leu Lys Val Ser Ser Thr Ala Val

725 730 735

Arg Thr Gln His Thr Thr Thr Arg Pro Val Pro Asp Thr Ser Arg Leu

740 745 750

Pro Gly Ala Thr Pro Gly Leu Thr Thr Val Glu Ile Val Thr Met Ser

755 760 765

His Gln Ala Leu Gly Asp Val Ala Gly Arg Gly Asn Glu Lys Lys Pro

770 775 780

Ser Ser Val Arg Asp Tyr Lys Asp Asp Asp Asp Lys His His His His

785 790 795 800

His His

<210> 8

<211> 331

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> shortened form of the LDLR ectodomain fragment and hIgG1-Fc fusion protein (with pCSK9 binding activity)

: LDLR-sECD-Fc (hIgG 1) as detection reagent

<400> 8

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser

20 25 30

His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp

35 40 45

Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu Cys

50 55 60

Gln Asp Pro Asp Thr Cys Ser Gln Leu Cys Val Asn Leu Glu Gly Gly

65 70 75 80

Tyr Lys Cys Gln Cys Glu Glu Gly Phe Gln Leu Asp Pro His Thr Lys

85 90 95

Ala Cys Lys Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro

100 105 110

Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro

115 120 125

Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

130 135 140

Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn

145 150 155 160

Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

165 170 175

Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

180 185 190

Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

195 200 205

Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys

210 215 220

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

225 230 235 240

Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

245 250 255

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

260 265 270

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

275 280 285

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

290 295 300

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

305 310 315 320

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 9

<211> 294

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> more shortened version of the LDLR ectodomain fragment with hIgG1-Fc fusion protein (with binding activity to pCSK9

Sex): LDLR-ssECD-Fc (hIgG 1) as detection reagent

<400> 9

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Gly Thr Asn Glu Cys Leu Asp Asn Asn Gly Gly Cys Ser

20 25 30

His Val Cys Asn Asp Leu Lys Ile Gly Tyr Glu Cys Leu Cys Pro Asp

35 40 45

Gly Phe Gln Leu Val Ala Gln Arg Arg Cys Glu Asp Ile Asp Glu Pro

50 55 60

Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu

65 70 75 80

Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

85 90 95

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

100 105 110

Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly

115 120 125

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn

130 135 140

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

145 150 155 160

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro

165 170 175

Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

180 185 190

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn

195 200 205

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

210 215 220

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

225 230 235 240

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

245 250 255

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

260 265 270

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

275 280 285

Ser Leu Ser Pro Gly Lys

290

<210> 10

<211> 121

<212> PRT

<213> mice (Mus musculus)

<400> 10

Gln Val His Leu Gln Gln Ser Gly Ala Glu Leu Ala Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Lys Glu Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Tyr Asp Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Thr Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 11

<211> 112

<212> PRT

<213> mice (Mus musculus)

<400> 11

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Arg Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 12

<211> 5

<212> PRT

<213> mice (Mus musculus)

<400> 12

Asp Tyr Trp Met His

1 5

<210> 13

<211> 17

<212> PRT

<213> mice (Mus musculus)

<400> 13

Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe Lys

1 5 10 15

Asp

<210> 14

<211> 12

<212> PRT

<213> mice (Mus musculus)

<400> 14

Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val

1 5 10

<210> 15

<211> 17

<212> PRT

<213> mice (Mus musculus)

<400> 15

Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Phe Leu

1 5 10 15

Ala

<210> 16

<211> 7

<212> PRT

<213> mice (Mus musculus)

<400> 16

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 17

<211> 8

<212> PRT

<213> mice (Mus musculus)

<400> 17

Lys Gln Ser Phe Asn Leu Phe Thr

1 5

<210> 18

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> ch-001

<400> 18

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 19

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VH.1A

<400> 19

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 20

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VH.1B

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 21

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VH.1C

<400> 21

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 22

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VH.1D

<400> 22

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 23

<211> 121

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VH.1E

<400> 23

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Asn Asp Tyr

20 25 30

Trp Met His Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 24

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> ch-001 hVL.1 (CDR grafted)

<400> 24

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 25

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VL.1A

<400> 25

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 26

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VL.1B

<400> 26

Asp Ile Gln Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 27

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-VL.1C

<400> 27

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 28

<211> 451

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-4-IgG1

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 29

<211> 1413

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-4-IgG1

<400> 29

atggagtttg ggctgagctg gctttttctt gtcgcgattc ttaagggtgt ccagtgccag 60

gtgcagctgg tgcagagcgg cgctgaggtg aagaagcccg gagcgagcgt aaaggtgagc 120

tgcaaggcca gcggatacac cttcaccgac tactggatgc actgggtgag gcaggcccca 180

ggacagggcc tggagtggat gggctacatc aaccccagca gcggctttac caagtatcac 240

cagaacttca aagacagggt gaccatgacc agggacacca gcatcagcac cgcctacatg 300

gagctgagca ggctgaggag cgacgacacc gccgtgtact actgcgccag gcaatacgac 360

tacgacgagg actggtactt cgacgtgtgg ggccaaggaa ccaccgtgac tgtgagcagc 420

gcttcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc tccgggtaaa tga 1413

<210> 30

<211> 219

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-4-kappa

<400> 30

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Phe Asn Leu Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 31

<211> 726

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-4-kappa

<400> 31

atggacatgc gcgtgcccgc ccagctgctg ggcctgctgc tgctgtggtt ccccggctcg 60

cgatgcgaca tcgtgatgtc tcagagccca tctagcctga gcgccagcgt gggcgacagg 120

gtaaccatca cctgcaagag cagccaaagc ctgctgaaca gcaggacccg caagaacttc 180

ctggcttggt atcagcagaa gcccggcaag tctcccaagt tgctgatcta ctgggccagc 240

accagggaga gcggcgtgcc cgacaggttc agcggctccg gcagcggcac cgacttcacc 300

ctgaccatct ctagtctgca gcccgaggac ttcgccacct actactgcaa gcagagcttc 360

aatctgttca ccttcggcca gggcaccaag ctggagatca agcgtacggt ggctgcacca 420

tctgtcttca tcttcccgcc atctgatgag cagttgaaat ctggaactgc ctctgttgtg 480

tgcctgctga ataacttcta tcccagagag gccaaagtac agtggaaggt ggataacgcc 540

ctccaatcgg gtaactccca ggagagtgtc acagagcagg acagcaagga cagcacctac 600

agcctcagca gcaccctgac gctgagcaaa gcagactacg agaaacacaa agtctacgcc 660

tgcgaagtca cccatcaggg cctgagctcg cccgtcacaa agagcttcaa caggggagag 720

tgttga 726

<210> 32

<211> 451

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-4-IgG1-YTE heavy chain

<400> 32

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Phe Thr Lys Tyr His Gln Asn Phe

50 55 60

Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gln Tyr Asp Tyr Asp Glu Asp Trp Tyr Phe Asp Val Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr

245 250 255

Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 33

<211> 1413

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> h001-4-IgG1-YTE heavy chain

<400> 33

atggagtttg ggctgagctg gctttttctt gtcgcgattc ttaagggtgt ccagtgccag 60

gtgcagctgg tgcagagcgg cgctgaggtg aagaagcccg gagcgagcgt aaaggtgagc 120

tgcaaggcca gcggatacac cttcaccgac tactggatgc actgggtgag gcaggcccca 180

ggacagggcc tggagtggat gggctacatc aaccccagca gcggctttac caagtatcac 240

cagaacttca aagacagggt gaccatgacc agggacacca gcatcagcac cgcctacatg 300

gagctgagca ggctgaggag cgacgacacc gccgtgtact actgcgccag gcaatacgac 360

tacgacgagg actggtactt cgacgtgtgg ggccaaggaa ccaccgtgac tgtgagcagc 420

gcttcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tctacatcac ccgggagcct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1140

ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc tccgggtaaa tga 1413

Claims

1. A pharmaceutical composition comprising a PCSK-9 antibody or antigen-binding fragment thereof and a histidine buffer; wherein the PCSK-9 antibody or antigen-binding fragment thereof has the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and

2. The pharmaceutical composition of claim 1, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of 1mg/ml to 150mg/ml.

3. The pharmaceutical composition of claim 2, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of 30mg/ml to 100mg/ml.

4. The pharmaceutical composition of claim 2, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of 45 to 55mg/ml.

5. The pharmaceutical composition of claim 2, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of 50mg/ml.

6. The pharmaceutical composition of claim 1, wherein the pH of the pharmaceutical composition is 5.5 to 6.5.

7. The pharmaceutical composition of claim 6, wherein the pH of the pharmaceutical composition is from 6.0 to 6.5.

8. The pharmaceutical composition of claim 6, wherein the pH of the pharmaceutical composition is 6.0.

9. The pharmaceutical composition of claim 1, wherein the histidine buffer is a histidine-hydrochloride buffer.

10. The pharmaceutical composition of claim 1, wherein the histidine buffer concentration is 5mM to 30mM.

11. The pharmaceutical composition of claim 10, wherein the histidine buffer concentration is 5mM to 20mM.

12. The pharmaceutical composition of claim 10, wherein the histidine buffer concentration is 5mM to 15mM.

13. The pharmaceutical composition of claim 10, wherein the histidine buffer concentration is 10mM.

14. The pharmaceutical composition of claim 1, further comprising a disaccharide.

15. The pharmaceutical composition of claim 14, wherein the disaccharide is selected from trehalose or sucrose.

16. The pharmaceutical composition of claim 14, wherein the disaccharide is sucrose.

17. The pharmaceutical composition of claim 14, wherein the disaccharide concentration is 10mg/ml to 75mg/ml.

18. The pharmaceutical composition of claim 17, wherein the disaccharide concentration is 20mg/ml to 40mg/ml.

19. The pharmaceutical composition of claim 17, wherein the disaccharide concentration is 25mg/ml.

20. The pharmaceutical composition of claim 1, further comprising a surfactant.

21. The pharmaceutical composition of claim 20, wherein the surfactant is a polysorbate.

22. The pharmaceutical composition of claim 20, wherein the surfactant is polysorbate 80.

23. The pharmaceutical composition of claim 20, wherein the concentration of surfactant is 0.05mg/ml to 0.6mg/ml.

24. The pharmaceutical composition of claim 23, wherein the concentration of the surfactant is 0.1mg/ml to 0.4mg/ml.

25. The pharmaceutical composition of claim 23, wherein the concentration of the surfactant is 0.1mg/ml to 0.3mg/ml.

26. The pharmaceutical composition of claim 23, wherein the concentration of the surfactant is 0.2mg/ml.

27. The pharmaceutical composition of claim 1, wherein the PCSK-9 antibody or antigen-binding fragment thereof has a heavy chain variable region selected from the group consisting of the heavy chain variable regions shown in SEQ ID NOs 18-23 and a light chain variable region selected from the group consisting of the light chain variable regions shown in SEQ ID NOs 24-27.

28. The pharmaceutical composition of claim 1, wherein the PCSK-9 antibody has a light chain amino acid sequence of SEQ ID NO:30, the heavy chain amino acid sequence of the PCSK-9 antibody is shown as SEQ ID NO:28 or 32.

29. A method of preparing the pharmaceutical composition of any one of claims 1 to 28, comprising: a step of replacing a PCSK-9 antibody or an antigen-binding fragment stock solution thereof by a buffer solution, wherein the buffer solution is histidine buffer solution, the concentration of the buffer solution is 5mM to 30mM, and the pH of the buffer solution is 6.0 to 6.5;

Wherein the PCSK-9 antibody or antigen-binding fragment thereof has the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and

30. The method of claim 29, further comprising the step of adding sucrose and polysorbate 80 to the solution obtained in the displacing step, and sizing the solution with histidine buffer at a concentration of 10mM to 20mM and at a pH of 6.0 to 6.5.

31. A method of preparing a lyophilized formulation comprising a PCSK-9 antibody comprising the step of lyophilizing the pharmaceutical composition of any one of claims 1 to 28;

32. A method of preparing a lyophilized formulation comprising a PCSK-9 antibody of claim 31 wherein the lyophilization comprises the steps of prefreezing, primary drying, and secondary drying in sequence.

33. A lyophilized formulation comprising a PCSK-9 antibody prepared by the process of claim 31 or 32.

34. A method for preparing a reconstitution solution containing a PCSK-9 antibody, comprising the step of reconstituting the lyophilized formulation of claim 33 with a solvent that is water for injection.

35. A reconstituted solution containing PCSK-9 antibody prepared by the method of claim 34: comprising a PCSK-9 antibody or antigen-binding fragment thereof, and a histidine buffer; wherein the PCSK-9 antibody or antigen-binding fragment thereof has the HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and

36. The PCSK-9 antibody-containing reconstituted solution of claim 35, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of 120mg/ml to 200mg/ml.

37. The PCSK-9 antibody-containing reconstituted solution of claim 36, wherein the PCSK-9 antibody or antigen-binding fragment thereof is at a concentration of 150mg/ml.

38. The PCSK-9 antibody-containing reconstitution solution of claim 35 wherein the pH of the reconstitution solution is between 6.0-6.5.

39. The PCSK-9 antibody-containing reconstitution solution of claim 38 wherein the pH of the reconstitution solution is 6.3.

40. The PCSK-9 antibody-containing reconstitution solution of claim 35, wherein the histidine buffer concentration is 15mM to 45mM.

41. The PCSK-9 antibody-containing reconstitution solution of claim 40, wherein the histidine buffer is at a concentration of 30mM.

42. The PCSK-9 antibody-containing reconstituted solution of claim 35 further comprising a disaccharide.

43. The PCSK-9 antibody-containing reconstitution solution of claim 42, wherein the disaccharide is selected from trehalose or sucrose.

44. The PCSK-9 antibody-containing reconstitution solution of claim 42, wherein the disaccharide is sucrose.

45. The PCSK-9 antibody-containing reconstituted solution of claim 42, wherein the disaccharide concentration is 55mg/ml to 95mg/ml.

46. The PCSK-9 antibody-containing reconstituted solution of claim 45, wherein the disaccharide concentration is 75mg/ml.

47. The PCSK-9 antibody-containing reconstituted solution of claim 35, further comprising a surfactant.

48. The PCSK-9 antibody-containing reconstitution solution of claim 47, wherein the surfactant is a polysorbate.

49. The PCSK-9 antibody-containing reconstitution solution of claim 47, wherein the surfactant is polysorbate 80.

50. The PCSK-9 antibody-containing reconstituted solution of claim 47, wherein the concentration of surfactant is 0.4mg/ml to 0.8mg/ml.

51. The PCSK-9 antibody-containing reconstituted solution of claim 50, wherein the concentration of surfactant is 0.6mg/ml.

52. An article of manufacture comprising a container containing a pharmaceutical composition according to any one of claims 1 to 28 or a lyophilized formulation according to claim 33 or a reconstituted solution of a lyophilized formulation according to any one of claims 35 to 51.